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Screening of biogenic amines production by bacteria isolated from "pecorino" cheese
No full textBiogenic amines (BA) are naturally present in many foods and beverages, especially fermented ones. Due to their toxicity, high levels of BAs in food can be a health risk. In cheese the most abundant amine is tyramine (TYR) which is the main cause of the so-called “cheese reaction”. BAs presence in food is mainly caused by aminoacids decarboxylating bacteria.
Aim of the study is toevaluate BAs in vitro production by bacteria isolated from a medium-ripened “pecorino” cheese.Materials and Methods: 72 strains of potential BAs producers (Enterococci (Ec), Enterobacteriaceae (Eb) and mesophilic lactobacilli (Lb)) were isolated during the cheesemaking and the ripening phases of a “pecorino” cheese, manufactured in a dairy factory in Tuscany. Ec (34) and Lb (28) were identified by PCR [1,2,3,4], Eb (10) were identified by API 20E kit (Biomérieux). After 72h incubation in a decarboxylase broth added with 1% w/v of the precursor aminoacids, the production of 7 BAs (triptamine, 2-phenylethylamine (2PHE), putrescine (PUT), cadaverine (CAD), histamine, TYR, spermidine, spermine) was quantified on the acid extract of the cultural medium by HPLC analysis as described in a previous study [5].
All the strain tested produced BAs although in varying degrees. Overall our data on BAs production by the different microbial groups agree with previous studies [6,7,8,9]. Eb were confirmed as good PUT and CAD producers, both for number of decarboxylating strains (100% and 90% respectively) and for BAs concentrations (on average 341 and 785 μg/ml, respectively). All Ec strains produced TYR, with very high mean amounts (1608μg/ml), and many of them gave not negligible 2PHE, PUT and CAD production (on average 184, 121 and 146 μg/ml, respectively). These data agree with Ladero et al. [10] hypothesis that TYR production is genomic trait of E. faecium and E. faecalis species. On the other hand the same Authors speculated that PUT is a genomic trait of E. faecalis, while in our study 50% of the tested strains of this species showed no detectable PUT production. Indeed, other Authors reported PUT-negative strains of E. faecalis [6,9,11]. Strains that produce high amounts of BAs, although with a low prevalence, when growing to high cell counts can substantially contribute to BAs formation [9]. In view of this, it is noteworthy that, although our data confirm that Lb are overall not good BA producers, few Lb strains, isolated from pasteurized milk cheeses, produced very high amounts of TYR (810, 1766 and 1959 μg/ml).
BA presence in cheeses is not solely associated with undesired bacterial groups (Eb, Ec), but technologically useful microorganisms, like Lb, could play a role in TYR accumulation in ripened cheeses, especially considering the high Lb loads reached and maintained throughout the ripening period
Detection of genes encoding for enterotoxins, TSST-1, and biofilm production in coagulase-negative staphylococci from bovine bulk tank milk
No full textCoagulase-negative staphylococci (CNS) represent common skin commensals
in humans and other mammals. They are opportunistic microorganisms and, in
dairy production, are considered as minor pathogens, often associated to subclinical
mastitis. It has been recently demonstrated that CNS can produce Staphylococcus
aureus superantigens, as well as biofilm. In our study, 74 CNS were isolated from
Tuscan (Central Italy) bovine bulk tank milk and phenotypically identified as
Staphylococcus xylosus (28.4 %), Staphylococcus chromogenes (20.3 %),
Staphylococcus sciuri (12.2 %), Staphylococcus hominis (9.4 %), Staphylococcus
haemolyticus (6.8 %), Staphylococcus capitis (5.4 %), Staphylococcus cohnii ssp.
cohnii (4.1 %), Staphylococcus saprophyticus (4.1 %), and Staphylococcus simulans
(4.1 %). Moreover, one isolate (1.3 %) was detected for each of the following species:
Staphylococcus caprae, Staphylococcus epidermidis, Staphylococcus lentus, and
Staphylococcus warneri. All isolates were screened by PCR for the presence of genes
encoding classical enterotoxins (sea-see), toxic shock syndrome toxin-1 (tst), and
biofilm (icaA/D). Forty isolates (54.1 %) presented at least one gene encoding for
enterotoxin production, 15 (20.3 %) harbored two or more genes in association. The
most frequently detected gene was sea (41.9 %), followed by sec-1 (25.7 %). None of
the tested isolates presented genes encoding for enterotoxin B or D. Fifteen isolates
(20.3 %) presented genes encoding for biofilm production. Among these, 11 isolates
also harbored genes encoding for enterotoxin production. Our results suggest that
enterotoxigenic CNS could represent a potential reservoir of pathogenicity factors,
which could substantially contribute to enhance S. aureus virulence when present in
milk and milk products. Further investigations are required for the evaluation of
enterotoxins and biofilm production
Detection of Mycobacterium avium subsp. paratuberculosis in cheeses from small ruminants in Tuscany
Full text linkParatuberculosis is an infectious disease which affects mainly domestic and wild ruminants caused by Mycobacterium avium subsp. paratuberculosis (Map). Map has been associated with human diseases like Crohn disease, type-1 diabetes, sarcoidosis, multiple sclerosis and Hashimoto's thyroiditis. The aim of this study was to determine the level of Map positivity of cheeses produced in Tuscany (Italy) as an indication of human exposure to the specific pathogen. Sampling was focused on artisanal cheeses produced without commercial starter culture from raw sheep or goat milk, on small-scale farms.Samples were tested by quantitative PCR (qPCR) and culture. Map DNA was detected in 4/7 (57.14%) goat, and in 14/25 (56%) sheep cheeses by qPCR, whereas cultivation produced a positive result in only one case. This corresponded to a goat cheese that had also reacted positively by qPCR and yielded a viable Type S (sheep) strain of Map. The Map load of the tested samples based on qPCR ranged from 6×10 to 1.8×104Map cells/g of cheese. The results indicate on average 56.57% and 66.6% positivity of cheese samples and farms, respectively. Hence, the type of cheeses that were analyzed within the context of this study seem to constitute a considerable source of human exposure to Map; although the question remains of whether the Map cells were present in a viable form, since positive results were almost exclusively recorded by qPCR
Is Leptospira able to survive in raw milk? Study on the inactivation at different storage times and temperatures
No full textThe consumption of raw milk is currently increasing due to several beneficial aspects, such as nutritional qualities, taste and health benefits. However, some Authors highlight the potential risk associated with raw milk consumption. While the absence of some pathogen microorganisms is set by the regional regulation DGR 381/2007, for other microorganisms, such as Leptospira, no recommendations are provided. Leptospira is not ascribed among classical milk pathogens, however, it can potentially be present in raw milk. The aim of this study was to evaluate the survival in raw milk of 6 serovars of Leptospira after storage at different temperatures (4°C +/- 2°C, 20°C +/- 2°C and 30°C +/- 2°C) for different incubation times (20 minutes, 45 minutes, 1 hour and 1 hour and 30 minutes), in order to determine the potential risk for consumers. Moreover, the immediate effect of bovine, goat and donkey raw milk on tested Leptospira serovars was visually evaluated. After incubation, all samples were subcultured in EMJH and incubated aerobically at 30 °C for 3 months. All inoculated media were weekly examined by dark-field microscope in order to assess Leptospira survival. Extemporary observation of strains’ behavior in milk allowed to detect an almost immediate motility loss and no leptospires were detected by microscopic observations carried out weekly during the trial period. According to our results, it could be possible to exclude raw milk as a source of Leptospira infection for consumers
Detection of Mycobacterium avium subsp paratuberculosis in a sheep flock in Tuscany
Full text linkParatuberculosis is an infectious disease which affects ruminants. In this work, a sheep flock in Garfagnana district (Tuscany, Italy) was examined by agar gel immune-diffusion (AGID) tests, culture, and PCR from feces, milk, tissue samples, and cheeses. At the first AGID test, 7/280 (2.5 %) animals were positive. From these animals, feces and milk samples were collected: 4/7 feces (57.14 %) and 2/7 milk samples (28.57 %) were positive to culture and 7/7 (100 %) feces samples and 1/7 milk samples (14.28 %) were positive to PCR; 2/4 (50 %) cheeses ripened for 14 days and 1/3 (33.33 %) cheese ripened for 20 days were positive to PCR, from which no viable microorganisms were isolated. Then, the AGID-positive animals were slaughtered and tissue samples were taken from one sheep with PCR-positive feces and milk: the liver, intestine, mesenteric lymph nodes, but not the spleen and mammary lymph nodes were positive to culture; all these samples were instead PCR-positive. After 1 year, a second AGID survey was performed on the remaining animals: 6/244 (2.45 %) subjects were positive to this test. Data obtained revealed the presence of paratuberculosis in a sheep population in Garfagnan
PHENOTYPICAL CHARACTERIZATION OF STAPHYLOCOCCUS AUREUS SEA POSITIVE STRAINS ISOLATED FROM RAW MILK: ENTEROTOXIN A PRODUCTION AND ANTIBIOTIC-RESISTANCE
No full textATTI del LXVIII CONVEGNO SISVE
The presence of Leptospira in coypus (Myocastor coypus) and rats (Rattus norvegicus) living in a protected wetland in Tuscany (Italy)
Full text linkFrom September 2009 to February 2011, 122 coypus (Myocastor coypus) and 74 rats (Rattus norvegicus) were captured employing cage traps in a protected wetland in Tuscany (central Italy). Blood serum samples were collected from the animals and successively examined by the microagglutination test for several serovars of Leptospira: Bratislava, Ballum, Bataviae, Grippothyphosa, Icterohaemorrhagiae, Copenhageni, Mini, Pomona, Zanoni, Sejroe, Hardjo and Tarassovi. Kidney samples were collected from each animal and tested by bacteriological methods and submitted to Polymerase Chain Reaction. Thirty-four (27.87 %) coypu sera were positive to Leptospira interrogans serovar Bratislava, with antibody titers ranging from 1:100 to 1:400; no strain was isolated from coypu by bacteriological examination, while 12 (9.83 %) subjects were positive to PCR. All rats resulted seronegative; thirty-seven (50 %) Leptospira strains were isolated from rat kidneys; 30 were classifi ed as Leptospira interrogans serovar Icterohaemorrhagiae and 7 as Leptospira interrogans serovar Ballum by the cross-agglutination test. Forty-fi ve (60.81 %) rats resulted positive to PCR: 37 subjects positive to bacteriological examination and there were eight from which no strain were isolated from kidneys. These results would seem to suggest the minor zooepidemiological role of coypu in leptospirosis, which is widespread according to literature
EFFETTO DELL’ADDIZIONE DI POLLINE D’API BIOLOGICO ESSICCATO SULLO SVILUPPO DI MICRORGANISMI PATOGENI IN LATTE
Full text linkLo scopo del presente lavoro è stato quello di valutare l'effetto dell'addizione
di una sospensione di polline d'api biologico essiccato sullo sviluppo in latte di alcuni
microrganismi patogeni. Il polline utilizzato è stato raccolto da api allevate in provincia di
Lucca (Toscana, Italia) nell’anno di vegetazione 2013 ed è stato utilizzato per preparare
una sospensione da addizionare in ragione dell’ 1%, 2%, 4% (v/v) a Skim Milk inoculato con
ceppi patogeni di referenza in cariche pari a 106 UFC/mL. I ceppi indagati sono stati
Enterococcus faecalis ATCC 19433, Staphylococcus aureus ATCC 6539, Listeria monocytogenes
ATCC 7644, Salmonella enterica sv. Thyphimurium ATCC 14028. Lo sviluppo dei microrganismi
è stato monitorato dopo 24 e 48 ore dall’inoculo in campioni mantenuti a temperatura
di refrigerazione e a temperatura ottimale di crescita. E’ stato inoltre valutato il profilo
microbiologico del polline impiegato. Il polline utilizzato presentava una carica batterica
mesofila pari a 4,9 log UFC/g, con cariche in Enterobacteriaceae e Lactobacillus spp. pari
a 3,7 log UFC/g e 4,9 log UFC/g, rispettivamente. Muffe e lieviti presentavano una carica
di 5,5 log UFC/g. Variazioni nelle conte in latte sono state rilevate solamente dopo incubazione
a temperatura ottimale di sviluppo. A 48 h di incubazione E. faecalis in latte al 4% di polline
ha mostrato una diminuzione delle cariche pari a 1 log UFC/mL; S. aureus è diminuito di
2 log ufc/ml in latte al 2% e 4% di polline; L. monocytogenes è diminuita di quasi 4 log
UFC/mL, sempre in latte al 4% di polline. Infine, S. enterica ha mostrato ben 6 log UFC/mL
di diminuzione dopo 48 ore in latte al 4% di polline. Analisi ulteriori sono necessarie al fine
di valutare in quale entità le diminuzioni riscontrate siano dovute ai composti antimicrobici
presenti nel polline o piuttosto ad un effetto di competizione biologica esercitato dalla
microflora naturalmente presente nel polline
DOES THE ADDITION OF DONKEY MILK INHIBIT THE REPLICATION OF PATHOGEN MICROORGANISMS IN GOAT MILK AT REFRIGERATED CONDITION?
No full textCurrently, donkey milk is receiving an increasing attention from consumers
and research community because of its several beneficial aspects, such as a poor
allergenic nature and a remarkable antimicrobial compound content. In this study, we
evaluated the growth rate of Staphylococcus aureus ATCC 6538, Listeria
monocytogenes ATCC 7644TM, Campylobacter jejuni ATCC 33291, and
Pseudomonas aeruginosa ATCC 27853 at refrigeration conditions (4±2 °C) in goat
milk added with different percentages of donkey milk (1, 2.5, 5, 10% v/v) along 6 days
of storage; furthermore, donkey milk and goat milk samples were employed as controls.
Lysozyme content of donkey milk was determined and ranged from 1 mg.mL−1 up to
2 mg.mL−1. An inhibited growth rate in donkey milk samples was observed during
storage for S. aureus, L. monocytogenes, and C. jejuni, with a growth decrease of 0.61
log CFU.mL−1, 5.55 log CFU.mL−1, and 1.72 log CFU.mL−1, respectively. These data
confirm the antibacterial activity of donkey milk against Gram-positive and Gramnegative
microorganisms; however, microbial growth rates in milk mixtures and goat
milk were comparable, with no significant increase in antibacterial activity due to
donkey milk addition. Considering a potential employment of donkey milk in dairy
products, further studies should be performed in order to detect the optimal balance of
milk mixtures in terms of caseins and antimicrobial molecules amounts
Occurrence of Clostridium spp. in ewe’s milk: enumeration and identification of isolates
No full textThe presence of gas-producing clostridia in ewe’s milk can lead to the occurrence of late-blowing defects in cheeses. However, data on this aspect are limited. In the present study, using the most probable number (MPN) method, clostridial spores were enumerated in 527 ewe’s milk samples collected in the Grosseto Province (Tuscany, Italy) from autumn 2014 to summer 2015. In addition, using polymerase chain reaction (PCR), we identified the species most frequently involved in lateblowing defects in cheese (Clostridium tyrobutyricum, Clostridium butyricum, Clostridium beijerinckii, and Clostridium sporogenes), and of Clostridium perfringens. Gas-producing clostridial spores were detected in 99% of samples. Spore concentrations ranged from 360 to more than 110,000 sporesIL−1. We observed that 86% of samples had a spore load higher than 1000 sporesIL−1. During autumn and summer, spore concentrations were significantly higher than in winter and spring (P < 0.001).A total of 222 isolates obtained from 77 MPN positive tubes from different milk samples were subjected to PCR. Colonies from 63/77 (82%) MPN positive tubes were taxonomically identified. Among the 63 PCR-positive samples, C. perfringens was the most frequently detected species (56%), followed by C. sporogenes (44%), C. tyrobutyricum (7.9%), C. butyricum (1.6%), and C. beijerinkii (1.6%). In addition, in 11% of the MPN positive tubes, at least two clostridial species were found to be present simultaneously. This work highlights the presence of clostridial spores in ovine milk from central Italy (Tuscany) and led to the identification of some of the clostridia species involved in such high spore load
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